This proposal studies cellular injury and repair in renal epithelial cells using a model called chemical anoxia to deplete the intracellular ATP stores. We have developed a reversible model of ATP depletion using chemical inhibitors of oxidative phosphorylation and glycolysis. Rotenone, an inhibitor of oxidative phosphorylation, and 2-deoxyglucose, ad competitive inhibitor of glycolysis, are added to renal epithelial cells grown on membrane filer supports. Following the application of the inhibitors, there is a rapid, reproducible fall in intracellular ATP. When a perfusion" media is added to the cells the intracellular ATP levels rapidly rise. At 2.5 hours following the addition or the perfusion media, the intracellular ATP levels are equivalent to controls. This new, reversible, chemical anoxia model will be used to study the nature of cellular injury and repair in two renal epithelial cell lines, MDCK (Madin-Darby canine kidney) and JTC (monkey proximal tubule cell line) in three ways. 1. The MDCK and JTC cell lines will be subjected to ATP depletion followed by restoration of their intracellular ATP reserves. This will allow us to test the hypothesis that renal cell recapitulate their normal developmental steps following the recovery from cellular injury (Bacallao and Fine, 1991). In these studies, the subcellular organization of the actin cytoskeleton, focal adherens, tight junction and adherens junctions will be examined by confocal laser scanning fluorescence microscopy. In addition we will study the recovery of the tight junction barrier function (Mandel, Bacallao and Zampighi, Nature, 1993) by measuring the transepithelial resistance across the epithelial monolayer at various times after recovery from chemical anoxia. 2. We will also test the hypothesis that cellular injury caused by chemical anoxia disrupts the accuracy of the protein sorting machinery of renal epithelial cells. This leads to loss in functional polarity. We suggest that the altered polarity of the injured cells will not be corrected until the protein sorting machinery is repaired. This hypothesis will be tested by quantifying the kinetics and stoichiometry of membrane assembly for a variety of endogenous membrane proteins. These studies may uncover significant different in the way proteins are sorted, especially during the recovery from the injury. For example the protein targeting mechanism of cytoskeletal linked proteins may be very sensitive to the effects of chemical anoxia, while other plasma membrane proteins may exhibit no change in their polarized delivery. These studies could potentially delineate different pathways of protein sorting. 3. Additionally, we will examine the effects of chemical anoxia on the protein salvage pathway. This pathway identifies mis-sorted proteins, removes them from the plasma membrane and delivers the proteins to the correct plasma membrane domain. This pathway will be examined by low pH fusion of vesicular stomatitis virus G protein in the apical membrane of the cells and determining the kinetics of delivery to the basolateral membrane.